Transplantation of neural cells

ABSTRACT

Restoration or increase of inhibitory interneuron function in vivo is achieved by transplantation of MGE cells into the brain. Compositions containing MGE cells are provided as are uses to treat various diseases characterized by abnormal inhibitory interneuron function or in cases where increase inhibition may ameliorate neural circuits that are abnormally activated.

GOVERNMENT SUPPORT CLAUSE

This invention was made with government support under grant no. NS048528awarded by the National Institutes of Health. The government has certainrights in the invention.

FIELD

The present invention relates to transplantation of neural cells toincrease inhibitory neuron activity in brain. It relates in particularto treatment of disorders that would benefit from increased inhibitoryneuron activity—this could include, but is not limited to diseasecharacterized by loss of inhibitory neuron function—and to compositionsuseful therefor and further relates to treatment of human diseaseincluding epilepsy and Parkinson's disease.

BACKGROUND TO THE INVENTION

Many neural disorders are characterised by abnormal inhibitory neuronsignalling and, in particular a lack of the neuro-transmitterγ-aminobutyric acid (GABA), secreted by inhibitory neurons. GABA, ametabolite of glutamate, is an inhibitory neurotransmitter whichcounteracts the effects of excitatory neurotransmitters. Excitatoryneurotransmitters (typically acetylcholine, glutamate, or serotonin)open cation channels, causing an influx of Na⁺ that depolarises thepostsynaptic membrane toward the threshold potential for firing anaction potential and hence cause the propagation of a signal across thesynapse. Inhibitory neurotransmitters, by contrast, open either Cl⁻channels or K⁺ channels, and this suppresses firing by making it harderfor excitatory influences to depolarise the postsynaptic membrane.

Abnormal inhibitory function may contribute to symptoms of Parkinson'sDisease and is fundamental to the pathology of several other neuraldisorders including Huntington's Disease, Schizophrenia, autism, chronicpain and many forms of Epilepsy. Epilepsy, in common with most suchdisorders, has no known cure and is treated with a range of drugs aimedat managing the symptoms. Therefore Epilepsy and its treatment result ina severe degradation of quality of life, measured in days of activity,pain, depression, anxiety, reduced vitality and insufficient sleep orrest (similar to arthritis, heart problems, diabetes, and cancer).

Epilepsy affects 50,000,000 people worldwide and sufferers have amortality rate two to three times higher than that of the generalpopulation with the risk of sudden death being 24 times greater. Inaddition to personal suffering, epilepsy imposes an annual economicburden of $15.5 billion in the USA alone, in associated health carecosts and losses in employment, wages, and productivity. Therefore anyalternative or new therapy, especially one with the potential to becurative, would have very far reaching benefits.

Research aiming to enhance inhibitory neuron function by celltransplantation has focused on the use of multi-potent cells andimmortalised neurons that have been genetically engineered to produceGABA (Bosch et al., (2004) Exp Neural 190, 42-58; Thompson, (2005)Neuroscience 133, 1029-37).

In order for the grafted cells to effectively reach affected regions andfunctionally integrate, it is necessary that the cells migrate away fromthe site of the graft and intermix with the host cells establishinginhibitory synapses with local excitatory neurons. A lack of migratoryactivity of the transplanted cells has been a flaw of previous attemptsto derive new neural tissue from precursor cells, such as in the case ofembryonic stem cell (ES)-derived neurons (Wernig et al., (2004) JNeurosci 24, 5258-68; Ruschenschmidt et al., (2005) Epilepsia 46 Suppl5, 174-83) and genetically engineered GABA-producing cells (Bosch et al,supra.; Thompson, supra). ES-derived cells or other neural precursorstransplanted into postnatal brains do not migrate extensively but formclumps of graft-derived cells in, or near, the site of transplantation(Bosch et al., supra; Ruschenschmidt et al., supra; Thompson, supra) andthus their value as a therapy is restricted, since usage would requiremultiple graft sites and only a limited volume of brain parenchyma canbe modified. It is also unlikely that the grafted cells could beadequately positioned to effectively increase inhibition if the positionof their cell body is constrained to the site of transplantation.

During development, cells from the medial ganglionic eminence forminhibitory interneurons. Studies on MGE cells are contradictory. Onerecent study (Olsson M et al. Neuroscience 69(4) 1169-82 (1995))concluded that MGE cells have a relatively low migratory capacity,compared with other neural precursor cells, when transplanted into ahost brain and that they would not be able to cross regions of the brainaffected by neural disease, whereas the paper by Butt et al. (Neuron 48,591-604, 2005) reported rapid migration.

ES cells have been shown to produce differentiated neurons in a hostbrain and so appear to be an excellent prospect for restoration ofinhibitory neuron function in the diseased brain. However, ES-derivedtransplants also form a heterogenous population of cells—althoughroughly 14% of ES-derived cells grafted into the postnatal brain expressGAD67 (a marker of GABA-containing interneurons), another 44% exhibit aglutaminergic phenotype, and so would be likely to have an excitatoryfunction (the opposite to that desired), and an unknown number arepresumably astrocytes (Wernig et al., supra). Also transplantation ofES-derived progenitor cells in order to increase GABAergic activity ofthe brain is fundamentally flawed, not only because of the limitedmigratory capacity of the cells, as mentioned above, but also because,following transplantation, formation of tumours is a common problem(Wernig et al., supra; Ruschenschmidt et al, supra).

OBJECTS OF THE INVENTION

An object of the present invention is to provide increased inhibitoryneuron function in the brain, and another object is to ameliorate or atleast provide an alternative therapy for diseases characterized byabnormal inhibitory interneuron activity or function. In addition anobject of particular embodiments is to increase inhibition in caseswhere inhibitory interneuron function is normal, but excess excitationmay cause pathological symptoms. An object of specific embodiments ofthe invention is to treat disease by transplantation of cells and fortransplanted cells or their progeny to disperse through disease-affectedareas and differentiate into mature neurons expressing appropriateneurotransmitters or neuropeptides. These cells should functionallyintegrate and directly influence circuitry in the damaged host brain.Preferably, grafted cells should be able to disperse through theaffected area and differentiate into neurons that contribute torestoration (or modulation) of existing neural circuit deficits. Assuch, transplantation of neuronal precursors can then be used as atherapeutic strategy for brain repair or circuit modification in whichincrease of inhibitory neuron function is required. These cells can alsobe used as vehicles to deliver the expression of molecules for a widerange of disorders including, but not limited to, cancer, infectiousdiseases, neurodegenerative diseases, traumatic brain injury, andpsychiatric disorders.

SUMMARY OF THE INVENTION

According to the present invention there is provided a method ofincreasing inhibitory neuron activity in the host central nervous systemin a mammal, comprising transplanting MGE cells into the brain of thatmammal. In particular, the method is for modifying inhibition in thebrain, such as a diseased brain.

The invention also provides a method of delivery of an inhibitoryinterneuron into a first portion of a mammalian brain, comprisingtransplantation of MGE cells into a second portion of the brain, distalfrom the first.

The invention further provides a method of creating an inhibitoryinterneuron, comprising obtaining an MGE cell and treating that cell soas to create an inhibitory interneuron with potential for functionalintegration in the host CNS.

Compositions of the invention are provided, comprising isolated humanMGE cells in a carrier, suitable for transplantation into a human brain.

The invention still further provides use of an MGE cell in manufactureof a composition for increasing inhibitory interneuron activity in amammal.

The invention hence provides methods and compositions to increaseinhibitory interneuron function in the central nervous system and canprovide methods and compositions and uses for treatment of diseasecharacterised by abnormal inhibition, especially such diseases asepilepsy and in particular such diseases in humans. In addition, theinvention hence provides methods and compositions and uses for treatmentof disease characterised by abnormal excitation, which can becharacteristic of diskinesias or neuropathic pain, and in particularsuch diseases in humans.

The invention additionally provides in certain embodiments a method todeliver therapeutic molecules for the treatment of disease, specificallyby expressing these molecules in transplanted MGE cells so that they arethen expressed in the functional interneurons produced.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based upon the transplantation of MGE cellsinto adult or immature brain so as to form new, functional inhibitoryinterneurons that can restore or modify neural circuits. A first aspectof the invention is a method of enhancing inhibition in a mammal,comprising transplanting MGE cells into the brain of that mammal. Themethod is of use in diseased brain, in which such interneurons have beenfunctionally impaired, damaged or destroyed, and so the inventionadvantageously provides for restoring inhibitory interneuron function inthe brain. Diseases which may benefit from increased inhibitory functionin the CNS can thus be treated such as those characterised by abnormalexcitatory neuron function.

In use, an MGE cell is transplanted and forms or creates an inhibitoryinterneuron de novo in the brain. Typically a plurality of cells isused, forming a plurality of interneurons. In examples described in moredetail below, these are found to have dispersed from the location oftransplantation and to have differentiated from the original MGE cells.

A second aspect of the invention is a method of delivery of aninhibitory interneuron into a first portion of a mammalian brain,comprising transplantation of MGE cells into a second portion of thebrain, distal from the first. Migration and subsequent differentiationof the MGE cell delivers the functional interneuron. Lack of inhibitoryinterneuron circuitry is commonly seen across many areas of diseasedbrain, and it is an advantage that the invention comprisestransplantation into one location from which cells and progeny disperse,providing interneuron populations in many distal locations. It is hencenot necessary to transplant cells into multiple loci. The interneuroncan be genetically engineered to express a heterologous gene. In anexample, the interneurons expressed GFP and other cells of the inventioncan be modified to express other proteins to be delivered to the brain.

The interneuron can also be genetically engineered to express aheterologous gene of therapeutic value. For example, MGE cells could beused to deliver proteins selected from: proteins for combating CNSmalignancies; proteins for treatment of epilepsies, e.g. by modifyingspecific signalling pathways; proteins for treatment ofneurodegenerative disorders, e.g. Alzheimers, including molecules thatcontribute to the clearance of neurotoxic substances; and proteins fortreatment of neuropsychiatric disorders, e.g. autism and schizophrenia.

A further aspect of the invention is a method of creating an inhibitoryinterneuron, comprising obtaining an MGE cell and treating that cell soas to create an inhibitory interneuron. The inhibitory interneuron ispreferably part of a neural circuit in which it provides inhibitoryfeedback via secretion of inhibitory neurotransmitters such as GABA. Asuitable treatment is to transplant the cell into mammalian brain,especially diseased brain.

The invention is of application generally to mammals, and in particularwherein the mammal is selected from the group consisting of mouse, rat,human, livestock animals and domestic animals. Preferably, the mammal isa human and the invention provides compositions containing human cellsand methods and uses for treatment of human disease.

MGE cells are described in a number of reports. For use in the presentinvention MGE cells from a variety of different sources may be used. Thecells may be obtained from foetal or embryo brain, for example bydissection of tissue and then dissociation of cells to yield acomposition comprising dissociated cells. MGE cells may also be obtainedby differentiation of a neural stem cell. Thus a neural stem cell istreated so as to differentiate into an MGE cell. The neural stem cellmay be obtained directly from tissue of a patient. It may be obtained bydifferentiation of a pluripotent cell, such as an ES cell.

In an embodiment of the invention, MGE cells are transplanted into aregion of the brain selected from hippocampus, cerebral cortex,subthalamic nuclei, other thalamic or hypothalamic regions, cerebellum,striatum and spinal cord.

Preferably, the method is for treatment of disease and the patient brainbeing treated comprises one or more lesions, such as a region withdamaged or destroyed inhibitory interneurons, the patient typicallybeing a mammal, especially a human, having consequent reduced inhibitoryinterneuron activity, or abnormal excitatory activity.

In an example set out in more detail below, dissociated MGE cells areinjected into the brain, preferably in association with a carrier, thiscarrier preferably being an air-buffered cell culture media.

Methods described herein are suitable for treatment of a patientafflicted by a disease characterised by inadequate inhibitoryinterneuron activity or increased excitatory neuron function and suchdiseases include Epilepsy, Parkinson's disease, Huntington's disease,Schizophrenia and chronic pain.

A further aspect of the invention is a composition, comprising isolatedhuman MGE cells in a carrier, suitable for transplantation into a humanbrain. The composition can easily be loaded into a syringe foradministration to the recipient.

Various carriers are suitable for the purpose, including tissue culturemedium. Preferably the carrier would have an appropriate osmolarity andpH in order to maintain the viability of the cells. In a typicaladministration from about 10⁵ to 10⁷, preferably from about 3×10⁵ to3×10⁶, cells are used, generally in from 0.5 to 20 μl of medium, and ata concentration of from 5000 to 2×10⁶ cells/μl, preferably from 5×10⁴ to10⁶/μl. It will be appreciated by one of reasonable skill in the artthat the number of cells and cell density may be optimized per host(e.g., human) through routine experimentation.

Still further aspects of the invention lie in the use of an MGE cell inmanufacture of a composition for enhancing inhibition in a mammal, thecomposition being preferably for restoring inhibitory interneuronfunction or counteracting elevated excitatory neural function e.g. fortreatment of a disease characterised by inadequate inhibitoryinterneuron activity or over activity of excitatory neurons such asneuropathic pain. Another aspect is the use described for de novocreation of an inhibitory interneuron, in particular in a human.

Referring to specific embodiments of the invention such as are describedin detail below, transplanted cells are MGE cells, or have thecharacteristic phenotype of MGE cells. Following transplantation, thesecells contribute to the inhibitory neuron function of the host brain,integrating into the host's brain whilst not being tumorigenic. Themigration is generally found to be fairly rapid, typically 5-10 μm/hour,facilitating distribution of the cells and progeny neurons throughoutthe brain. This migration allows delivery of interneurons into regionsof the brain distinct from the site of transplantation, e.g.transplantation into the cerebral cortex can result in an inhibitoryinterneuron creation in the hippocampus. Transplanted cells may betracked following implantation using molecular markers (e.g. GFP).

Transplanted MGE-like precursors form differentiated interneurons in thehost's brain, adopting the morphology of inhibitory interneurons, andhave been found to have the ability to migrate across the lesions in thebrain which can occur in neural disease. Transplanted cells adopt thephenotype of inhibitory interneurons, such that they express moleculescharacteristic of mature inhibitory neurons, and are found to alterneural function within the host brain, preferably in a permanent manner.Preferably transplanted cells do not form cortical pyramidal neurons anddo not increase excitatory neuron activity in the brain, but cause a netincrease in inhibitory neuron function in the brain relative toexcitatory function. Transplanted cells hence are used to restoreinhibitory neural function to normal levels in diseases characterised bya lack of inhibitory neural function or pathological excitation. Thecells, after integration into the host brain, receive synaptic inputs.The cells, after integration into the host brain, also receiveexcitatory inputs.

An advantage of the invention is that, following transplantation of anMGE cell, there is migration of the cell and formation in situ of afunctioning inhibitory interneuron. As a result, and referring to theexamples subscribed herein, there is enhanced inhibitory interneuronactivity in the recipient due to formation of a functional inhibitoryinterneuron. This interneuron can be a replacement for one lost due todisease or could be an additional interneuron. This interneuron not onlyreceives synaptic inputs but also excitatory inputs. A consequence ofthe inhibitory outputs, that the cells are capable of producing, is anincrease in GABA mediated synaptic events in the vicinity of the MGEcell derived inhibitory neuron.

A further advantage is that the cells produce mature GABA-secretinginterneurons in situ and there is no need artificially to modifytransplanted cells so as to secrete GABA.

The invention can thus provide treatment for diseases, such as Epilepsyand other diseases discussed herein, where lack of inhibitoryinterneuron function and consequent over-activity or inadequateregulation of excitatory interneurons forms an underlying element to thedisease.

In an example of the invention discussed in more detail below, MGE cellsare obtained by mechanical disruption of a dissected portion of foetaland/or embryonic brain. MGE cells can thus be obtained fortransplantation into humans. It is preferred that any MGEcell-containing composition is relatively pure in that othercontaminating cells are substantially removed. In certain embodiments ofthe invention the cellular component of the MGE cell-containingcomposition comprises at least 85%, at least 90%, or at least 95% MGEcells. In some embodiments at least 98% of the cells are MGE cells.

In the art, drug-based therapies are known in which levels ofneurotransmitters such GABA in the brain are increased, sometimesleading to a generalised increase in inhibitory activity. A feature ofthe present invention is that inhibitory interneurons are formed de novoand in situ in the brain, typically forming functional synapses so as torestore neural circuits—in the case, for example, of Epilepsy byrestoring normal regulation of neural circuits with formation aninhibitory interneuron. Rather than simply treating a symptom of thesediseases, an advantage of the invention is that an underlying cause ofthe disease is directly addressed. It also provides a method to targetinhibition to an area restricted by the migration of grafted cells. Thisis in contrast to therapies that increase inhibition throughout thenervous system.

DESCRIPTION OF THE DRAWINGS

The invention is now described in the following specific examples, withreference to the accompanying drawings, in which:

FIG. 1 shows MGE cells migrate rapidly following graft and so distributethroughout the host's brain;

FIG. 2 shows MGE cells distributed throughout the host's brain adopt amature interneuron morphology;

FIG. 3 shows integrated MGE cells in the somatosensory and cingulatecortex express molecules that characterize interneurons;

FIG. 4 shows grafted MGE derived cells are present in the dentate gyrusof the hippocampus 60 DAT.

FIG. 5 shows integrated MGE-derived cells function in a mannercharacteristic of inhibitory interneurons;

FIG. 6 shows recording configuration for analysis of inhibitory currentin host brain;

FIG. 7: shows MGE grafted cells alter synaptic function in the hostbrain;

FIG. 8 shows synaptic inhibitory current is increased in the hippocampusfrom grafted mice;

FIG. 9 shows glutamatergic synaptic excitation is not altered inneocortex of MGE grafted mice; and

FIG. 10 shows cortical brain slices prepared from Dlx mutant micetransplanted with MGE progenitor cells early in development (P0-P2)exhibit a level of inhibition (measured as spontaneous and miniatureIPSCs on postsynaptic pyramidal cell targets in regions containingMGE-GFP interneurons) that is comparable to that observed in control Dlxheterozygote mice.

DETAILED DESCRIPTION OF DRAWINGS

In more detail, FIG. 1 shows distribution of MGE derived cells 3 daysafter transplantation into neocortex and striatum. (A) MGE derived cellswere detected by immunohistochemistry against GFP. Serial sections wereutilized to determine the position of labelled cells. Notice the widedistribution throughout neocortex, striatum, and hippocampus. (B) Highmagnification of area in A showing MGE cells moving away from injectionsite (*). (C) Detail of a typical MGE migrating cell. (D) Distributionof grafted cells 3 and 60 DAT; number of cells/distance of serialsections. Scale bar in A: 1 mm; B: 250 μm; D: 25 μm. F, frontal; D,Dorsal; L, Lateral;

FIG. 2 shows acquisition and distribution of mature interneuronmorphology at 60 DAT. (A) Camera lucida maps indicating the position ofMGE graft-derived cells at three rostrocaudal levels aftertransplantation into neocortex (Ctx), hippocampus (Hp), and striatum(St). (B) Detection of grafted cells by immunohistochemistry against GFPin the ipsilateral somatosensory cortex. Note the wide distribution ofgrafted cells in multiple cortical layers. Compare the dark backgroundin layers I-II and V of the injected hemisphere (B) versus thecontralateral hemisphere (C). (E-K) GFP detection byimmunohistochemistry provides a Golgi-like staining of grafted cells.MGE-derived cells in cortex differentiated into neurons presentingtypical morphology of interneuron subtypes e.g., bitufted or bipolarcells (E), chandelier cells (F) with synaptic boutons resemblingcandlesticks (arrowheads), basket cells (H), neurons with small body(I), and multipolar cells (J). In hippocampus, grafted cells accumulatedin CA1 (D) and dentate gyrus (G). In striatum, the vast majority ofcells differentiated into medium aspiny interneuron (K). Scale bars inB, C, D, F, H and I: 100 μm; E, G, J and K: 50 μm;

FIG. 3 shows molecular characterization of MGE graft-derived cells insomatosensory (A-F, J-O), and cingulate cortex (G-I), 60 DAT.Immunohistochemical co-localization of grafted GFP⁺ cells with GABA,Parvalbumin (PV), Calretinin (CR), Somatostatin (SOM), andNeuropeptide-Y (NP-Y). Arrowheads show double positive cells for GFP andspecific marker. Scale bar 50 μm for A-O;

FIG. 4 shows grafted MGE derived cells in the dentate gyrus of thehippocampus 60 DAT. Immunohistochemical co-localization of MGE derivedcells expressing GFP with GABA (A-C), Parvalbumin (PV) (D-F), andSomatostatin (SOM) (G I). Arrowheads show double positive cells. Scalebar 100 μm for A-I;

FIG. 5 shows MGE-derived cells exhibit interneuronal firing properties.(A) IR-DIC image overlayed with an epifluorescence image of an acutecoronal slice (4 weeks post-grafting) containing GFP⁺ MGE-derived cells;epifluorescence image at right of a cell filled with Alexa red duringthe patch recording. (B) Membrane potential of the GFP⁺ cell shown inpanel A recorded under current clamp at the resting potential (˜−71 mV).Note the small degree of inward rectification with hyperpolarizingcurrent steps (200 ms) the lack of spike frequency adaptation with longdepolarizing current steps (1000 ms) typical of mature corticalinterneurons. (C) Graph of firing frequency of recorded GFP⁺ cells atdepolarizing step of 0.2 nA (n=14). Note the linear frequency-currentrelationship (inset graph);

FIG. 6 shows recording configuration for analysis of inhibitory currentin the host brain (A) Left panel shows a representative example of anacute coronal slice. Box indicates region in which electrophysiologicalrecordings were obtained. (B) Panel shows the acute coronal slice withGFP⁺ cells in Layers I-III visualized under IR-DIC and epifluorescencemicroscope. A recording was obtained from a pyramidal neuron (asterisk)in the vicinity of GFP⁺ cells. (C) Panel shows a higher magnification ofthe recording site with GFP⁺ MGE cells (green arrows) and a Luciferyellow filled pyramidal neuron (yellow asterisk);

FIG. 7 shows MGE grafted cells alter synaptic function in the hostbrain. (A) Sample traces of sIPSCs recorded from pyramidal cells(control brain and grafted brain); 4 weeks post-grafting. Note theincrease in IPSC amplitude and frequency for grafted animals vs.age-matched controls. (B) Cumulative data plots for all IPSC recordingsfrom control (light gray bars) and grafted (black bars) animals areshown. Recordings were made at 2, 3, and 4 weeks following grafting.Data represent 7-10 cells for each bar; data presented as mean±S.E.M.;significance taken as p<0.05 using one-way ANOVA. (C, D) Measurement ofthe total charge transfer for pyramidal cells from control and graftedbrain. Note the significant increase for grafted brains at 4 weeks. (E)Cumulative probability plots of sIPSCs inter-event intervals show higherfrequency values for grafted brains (p<0.05);

FIG. 8 shows synaptic inhibitory current is increased in the hippocampusfrom grafted mice. (A) Spontaneous IPSCs of hippocampal pyramidal cellsfrom control grafted mice with plots of frequency and amplitude ofsIPSCs of hippocampal pyramidal cells from control (light gray bars;n=10) and grafted mice (black bars; n=10). (B) Measurement of the totalcharge transfer of IPSCs recorded from CA1 hippocampal pyramidal cellsfrom control and grafted brain. Note the significant increase values forgrafted brains at 4 weeks. (C) Cumulative probability plots of sIPSCsinter-event intervals shown higher frequency values for grafted brains(p<0.05). Error bars indicate SEM.; *p<0.001; **p<0.05(ANOVA);

FIG. 9 shows glutamatergic synaptic excitation is not altered inneocortex and of MGE grafted mice. (A) Plots of all cortical pyramidalcells sampled for spontaneous EPSC data. sEPSC amplitude, decay-time andfrequency show no significant difference between controls (light graybars) and grafted (black bars) brains (B) Representative traces ofsEPSCs recorded from a GFP⁺ grafted cell at 4 weeks post-grafting.sEPSCs were abolished by application of CNQX and APV (bottom trace) (C)Sample of eEPSC recording from GFP⁺ grafted cells at different holdingpotentials showing the reversal membrane potential at 0 mV (see insetgraph); and

FIG. 10 shows cortical brain slices prepared from Dlx mutant micetransplanted with MGE progenitor cells early in development (P0-P2)exhibit a level of inhibition (measured as spontaneous and miniatureIPSCs on postsynaptic pyramidal cell targets in regions containingMGE-GFP interneurons) that is comparable to that observed in control Dlxheterozygote mice.

Example 1

MGE cells were transplanted from mice expressing green fluorescentprotein (GFP) into the postnatal brain. The time course of migration anddifferentiation of these neuronal precursors was determined. Also themolecular phenotype of transplanted MGE precursors was analysed usingantibodies directed against GABA, somatostatin (SOM) and neuropeptide Y(NPY). Using cortical slices from grafted animals we showed that MGE-GFPneurons exhibit intrinsic firing properties similar to fast-firingbasket-type cortical interneurons. Electrophysiological measurementsdemonstrate that MGE-derived neurons increase the level of GABA-mediatedsynaptic inhibition, and therefore appear to modify neocorticalinhibitory tone.

Materials and Methods

Tissue Dissection and Cell Dissociation.

Ventricular and subventricular layers from the anterior part of themedial ganglionic eminence, where a sulcus clearly divides medial andlateral ganglionic eminences, were dissected from E12.5-E13.5 embryonicGFP⁺ transgenic mice (Hadjantonakis et al., (1998) Mech Dev 76, 79-90).The day when the sperm plug was detected was considered E0.5. Borderingtissue between adjacent regions was discarded during dissection to avoidcontamination. Tissue explants were mechanically dissociated by repeatedpipetting through 200 μl yellow plastic tip (10-20 times). Dissociatedcells were washed with 1 ml of L-15 medium containing DNase I (10-100μg/ml) and pelleted by centrifugation (2 minutes, 800 g). Cells wereresuspended in 4-5 μl of L-15 medium and kept on ice until further use.

Transplantation.

Highly concentrated cell suspension (˜10⁶ cells/pi) was front-loadedinto beveled glass micropipettes (˜50 μm diameter) that were pre-filledwith mineral oil and L-15 medium. Micropipettes were connected to amicroinjector mounted on a stereotactic apparatus specially adapted forneonatal mice. 3-4 days old CD-1 mice (Charles River) were anesthetizedby exposure to −4° C. until pedal reflex was abolished. Anesthesia wasmaintained by performing surgery on a cold aluminum plate. 5×10⁴cells/mouse in a 50-100 nl volume were injected using a 45° inclinationangle and the following coordinates from Bregma: Striatum (3.3 mm A, 2.5mm L, 2.6 mm D); Cortex (2.2 mm A, 3.5 mm L, 1.2 mm D); Hippocampus (1.2mm A, 1.7 mm L, 2.0 mm D). For survival and migration distanceestimations, 5×10³ cells were grafted in a single point (2.5 mm A, 3.0mm L, 2.5-1.5 mm D). Grafted pups were returned to their mothers andanalyzed after 3 days, 1, 2, 3, 4 weeks and 3 months. All experimentalanimals were treated in accordance with UCSF Laboratory Animal ResearchCenter guidelines.

Immunostaining.

Animals were transcardially perfused with 4% paraformaldehyde atdifferent ages. Brains were removed, postfixed overnight in the samesolution, and sectioned coronally (50 μm) using a Vibratome. Floatingbrain sections were immunostained with the following antibodies: rabbitanti-GABA (1:2500, Sigma), mouse anti-parvalbumin (1:4000, Sigma) andrabbit anti-calretinin (1:4000, Swant Swiss Abs), rat anti-somatostatin(SOM) (1:500, Chemicon), rabbit anti-neuropeptide Y (1:5000,ImmunoStar), and mouse anti-GFP (1:200, Q-Biogene). The followingsecondary antibodies were used: cy3-conjugated donkey anti-mouse,cy3-conjugated donkey anti-rabbit, cy2-conjugated donkey anti-mouse andbiotin-conjugated donkey anti-mouse (1:400, all from JacksonImmunoResearch, PA). Sections were washed in PBS, blocked for 1 h in PBScontaining 10% donkey serum and 0.1% Triton X-100 at room temperature.Sections were then incubated overnight at 4° C. in primary antibodiesdiluted in PBS containing 10% donkey serum and 0.1% Triton X-100, thenwere washed three times in PBS and incubated with secondary antibodiesfor 1-2 h at room temperature in the dark. For GABA immunostaining,Triton X-100 was eliminated from the protocol. Biotinylated secondaryantibodies and ABC kit (Vector) were used for peroxidase reaction withdiaminobenzidine (DAB).

Cell Counts and Quantification.

Quantifications of cell bodies stained with immunohistochemistry or GFPwere counted on digitized images obtained with a DFC480 digital cameraand IM500/FW4000 image manager software (Leica Microsystems ImagingSolutions, Cambridge, UK) on a DM6000B microscope (Leica Microsystems,Wetzlar, Germany). Survival percentage of grafted cells was estimatedcounting all GFP⁺ cells in 10 coronal sections (300 μm apart, 1 sectionwith injection site, 4 forward to the injection, and 5 backward). Arepresentation of cell number vs. distance to injection site wasobtained on graph paper. Quantification of area under the graph wasestimated as total number of survived cells.

The percentage of grafted GFP⁺ cells expressing GABA, PV, CR, SOM or NPYafter transplantation was calculated in 3 coronal sections through eachof the following regions: somatosensory cortex, striatum andhippocampus. For somatosensory each section was 500 μm apart, usingstereotaxic coordinates (bregma levels +0.50 and −0.50 mm; Paxinos andFranklin, 2001); striatum sections were 400 μm apart, (bregma levels+1.60 and +0.80 mm); and for hippocampus, sections were 300 μm apart,(bregma levels—1.50 and −2.10 mm). At least 100 GFP⁺ cells (˜50 incortical layers II-IV, and ˜50 in layers V-VI, visualized using DAPI)were analyzed for each marker in each animal. Brains (n=5) were analyzedat 1, 3 and 6 months after transplantation. Statistical analysis wasperformed using the Student's t-test.

Quantifications of neuronal bodies stained by immunohistochemistry forinterneuron markers in grafted and contralateral hemispheres wereobtained as follows: In somatosensory cortex, 5 coronal sections (400 μmapart) per mouse between septum (bregma level+0.75 mm) and dorsalhippocampus (bregma level −1.25 mm) were selected. A 1 mm strip ofcortex from the white matter to pial surface was analyzed in eachsection (1.2 mm² each). In hippocampus, the numbers of positiveinterneurons in the hilus and CA1 areas were determined in 3 coronalsections (300 μm apart, between bregma levels—1.50 and −2.10 mm) permouse. In striatum, positive cells were counted in 3 coronal sections(400 μm apart, between bregma levels+1.60 and +0.80 mm) per mouse.Brains from at least 5 different grafted mice were counted and averaged.To compare results between grafted and contralateral hemispherestatistical analysis using the Student's t-test was applied andcontralateral results were referred as 100%. Results are presented asmean±SEM. Significance level was taken as p<0.05.

Electrophysiology.

Acute tissue slices were prepared from male or female CD-1 mice 2, 3,and 4 weeks after grafted with MGE cells or saline (control) as previousdescribed (Calcagnotto et al., (2002) J Neurosci 22, 7596-605).Whole-cell recordings were obtained from visually identified neurons(pyramidal cells and GFP⁺ cells) using an infrared differentialinterference contrast (IR-DIC) video microscopy system andepifluorescence microscopy (Molecular Devices). Intracellular patchpipette solution used for whole-cell voltage-clamp recordings to studyinhibitory postsynaptic current (IPSC) contained (in mM) 120Cs-gluconate, HEPES, 11 EGTA, 11 CsCl₂, 1 MgCl₂, 1.25 QX314, 2Na_(z)-ATP, 0.5 Na₂-GTP, (pH 7.25; 285-290 mOsm); for excitatorypostsynaptic current (EPSC) solution contained (in mM) 135 CsCl₂, 10NaCl, 2 MgCl₂, 10 HEPES, 10 EGTA, 2 Na₂ATP, 0.2 Na₂GTP, and 1.25 QX-314,adjusted to pH 7.2 with CsOH (285-290 mOsm). To isolate GABAergiccurrents, slices were perfused with nACSF containing 20 μM6-ciano-7-dinitroquinoxaline-2,3-dione (CNQX) and 50 μMd-(−)-2-amino-5-phosphonovaleric acid (D-APV) and IPSCs were recorded ata holding potential of 0 mV; for excitatory postsynaptic currents(EPSC), slices were perfused with nACSF containing 10 μM bicucullinemethiodide (BMI) and recorded currents at a holding potential of −75 mVunless otherwise noted. Miniature inhibitory synaptic currents (mIPSCs)were recorded in nACSF containing 1 μM tetrodotoxin (TTX). IPSCs/EPSCswere recorded on “aged-matched” pyramidal neurons (MGE graft-derived orsham-operated) either in the same slice or in a different one.Age-matched refers to slices obtained from mice within a three day timeperiod. Evoked currents were elicited using a monopolar electrode placedin the white matter. Pyramidal cells were filled with biocytin andanalyzed post hoc. To study the intrinsic firing properties of GFP⁺cells in current-clamp intracellular patch pipette solution contained(in mM) 120 KMeGluconate, 10 KCl, 1 MgCl₂, 0.025 CaCl₂, 10 HEPES, 0.2EGTA, 2 Mg-ATP, 0.2 Na-GTP, pH 7.2, (285-290 mOsm). Cells weredepolarized and hyperpolarized, via direct current injection (5-1000 ms,duration); cells were filled with Alexa red and analyzed post hoc.Voltage and current were recorded with an Axopatch 1D amplifier (AxonInstruments), and monitored with an oscilloscope and with pClamp 8.2software (Axon Instruments), running on a PC Pentium computer (DellComputer Company, Round Rock, Tex.). Whole-cell voltage-clamp data werelow-pass filtered at 1 kHz (−3 dB, 8-pole Bessel), digitally sampled at10 kHz. Whole-cell access resistance was carefully monitored throughoutthe recording and cells were rejected if values changed by more than 25%(or exceeded 20 MΩ); only recordings with stable series resistance of<20 MΩ were used for analysis (Mini Analysis 5.6.28 software;Synaptosoft, Decatur, Ga.). Results are presented as the mean±SEM. Tocompare results between different cell types, we used a one-way ANOVAwith significance level of p<0.05.

Results

Embryonic MGE Cells Grafted in Juvenile Brain Rapidly Disperse LongDistances.

To establish an efficient method for the transplantation and functionalassessment of MGE progenitors in a host brain, the MGE was dissectedfrom transgenic E12.5-E13.5 mice expressing green fluorescent protein(GFP) (Hadjantonakis et al., supra) GFP expression was used to track themigration and differentiation of grafted cells in live or fixed tissue.After mechanical dissociation, GFP⁺ MGE cells were loaded into a glassmicropipette and grafted into the neocortex and dorsal striatum in thebrain of postnatal day 3 or 4 (P3-P4) mice (Lois and Alvarez-Buylla,(1994) Science 264, 1145-8). Host animals were then sacrificed at 3days, 1, 2, 3 and 4 weeks post-grafting. Representative examples of theinjection sites and post-migratory behaviors of GFP⁺ cells are shown inFIGS. 1A and 2A.

Three days after transplantation (DAT) many GFP⁺ cells had migrated awayfrom the injection site (FIG. 1B) into most of the neocortex, striatumand hippocampus (FIG. 1A). Survival rate of grafted cells at this timepoint was 38.9±7.3% (n=10). At 3 DAT most GFP⁺ cells had the typicalmorphology of tangentially migrating interneurons, with asmall-elongated cell soma and a forked leading process (FIG. 1C). GFP⁺cells spread extensively around the injection site in all directions.Grafted cells covered a linear distance of 336±82 μm/day (n=20), with amaximum of 525 μm/day, analyzed 3 DAT; this speed of migration isgreater than reported in adults (˜120 μm/day) and similar to thatmeasured in vitro (280 μm/day on matrigel) (Wichterle et al., (1999) NatNeurosci 2, 461-6). A representation of cell number versus migrationdistances at 3 DAT results in a bell-shape curve (FIG. 1D). These datasuggest that cells did not have a strong preference for a particularmigratory route and disperse in all directions from the injection site.

Differentiation of Grafted MGE Cells in the Host Brain.

Analysis of grafted brains 7 DAT revealed a widespread distribution ofGFP⁺ MGE cells. At 7 DAT, most grafted cells no longer exhibited amigratory morphology; instead they had multiple processes and some cellshad a thin and longer axon-like process (data not shown). This indicatesthat initiation of differentiation of grafted MGE-derived neuronalprecursors occurs between three and seven days after transplantation.

Fourteen and 21 DAT, cells acquired progressively a more maturemorphology, showing larger and more elaborated dendritic trees withlonger axons. At 30 DAT, some GFP⁺ cells were more than 5 mm away frominjection site; their distribution was similar to that found at 3 DAT(FIGS. 1C & 2A). However, the survival percentage was reduced to19.9±3.9% (n=10). A similar level of survival, 21.2±4.1% (n=10), wasobserved at 90 DAT. The morphology of the grafted cells was studiedfollowing GFP immunohistochemistry, which provides Golgi-like staining.Two months after transplantation, GFP⁺ cells had elaborate dendritictrees extending profusely through cortical layers (FIG. 2). Axons andtheir presynaptic terminals could also be visualized (FIGS. 2B-C). Thusgrafted cells appeared to complete their differentiation intofunctionally integrated interneurons within one month aftertransplantation.

MGE-derived cells in the cortex differentiated into neurons withmorphologies of at least five different interneuron subtypes e.g.,bitufted or bipolar cells, chandelier cells, basket cells, neurons withsmall body, and multipolar cells (FIG. 2). For instance, some neuronsdisplayed synaptic buttons resembling arrays of candlesticks, suggestingthat they differentiated into chandelier cells (FIGS. 2B, E, F, H, I,J). In contrast, grafted cells in the striatum differentiate primarilyto medium aspiny interneurons (FIG. 2K), and in the hippocampus tointerneurons with morphologies typical for this region (basket,axo-axonic, and bistratified cells) (FIGS. 2D & G). None of theMGE-derived neurons exhibited morphological features of corticalpyramidal neurons e.g., triangular cell soma extending a thick spinyapical dendrite. Some immature oligodendrocytes were always noted aroundthe injection site; especially close to the corpus callosum, andoccasionally in the cortex where they were radially aligned (data notshown). GFP⁺ cells with an astrocytic morphology were not observed.Therefore, the MGE cells that we grafted are primarily committed to aninterneuronal lineage.

MGE-Derived Cells Exhibit Molecular Properties of Cortical Interneurons.

Recent studies suggest that MGE progenitors are the principal source ofcortical GABAergic interneurons (Lavdas et al., (1999) J Neurosci 19,7881-8; Sussel et al., (1999) Development 126, 3359-70; Anderson et al.,(2001) Development 128, 353-63; Wichterle et al., (2001) Development128, 3759-71). Interneurons can be classified into several subtypesbased on neurochemical markers, such as Ca²⁺-binding proteins(parvalbumin (PV), calbindin (CB), and calretinin (CR)), neuropeptides(e.g., somatostatin (SOM), neuropeptide Y (NPY), cholecystokinin (CCK),and vasoactive intestinal polypeptide (VIP)) (DeFelipe, (1993) CerebCortex 3, 273-89; Kubota et al., (1994) Brain Res 649, 159-73; DeFelipe,(1997) J Chem Neuroanat 14, 1-19; Gonchar and Burkhalter, (1997) CerebCortex 7, 347-58; DeFelipe, (2002) Frog Brain Res 136, 215-38), andrecording their physiological properties (Freund and Buzsaki, (1996)Hippocampus 6, 347-470; Cauli et al., (1997) J Neurosci 17, 3894-906;Gupta et al., (2000) Science 287, 273-8; Klausberger et al., (2003)Nature 421, 844-8). To evaluate the interneuronal phenotype andmolecular characteristics of transplanted MGE-GFP cells, we performed aseries of immunohistochemical studies 60 DAT. Double-immunofluorescencerevealed that approximately 65-70% of cortical GFP⁺ graft-derived cellsexpress GABA (FIG. 3; Table 1); a comparable level of GFP⁺ cells weredouble-labeled with an antibody against GAD67 (˜70%; data not shown).Subsets of the GFP⁺ neurons express NPY, SOM, PV, and CR (FIG. 3; Table1), at expression levels and in a distribution similar to those of thehost interneurons. Interestingly, SOM-expressing neurons were enrichedin layers I-II of the cortex, whereas CR positive cells were almostexclusively found in retrosplenial and cingulate cortex. This suggeststhat local environment contributes to the specification of someinterneuron sub-types.

MGE-derived cells were also immunopositive for these neurotransmittersand markers in the striatum and hippocampus (FIG. 4, Table 1). They weredistributed in the same areas that usually contain these types ofinterneurons. GFP⁺ cells were immuno-negative for antibodies to glialfibrillary acidic protein (GFAP), or choline acetyl transferase (ChAT),indicating that grafted cells did not differentiate into astrocytes orcholinergic neurons.

MGE-Derived Cells Exhibit Interneuronal Firing Properties.

To assess whether the MGE-derived cells had electrophysiologicalcharacteristics of cortical interneurons, GFP⁺ cells were targeted forwhole-cell current-clamp recording at 4 weeks post grafting. Diffusionof Alexa Red from the patch pipette permitted real-time confirmation ofcellular recording site (FIG. 5A). If MGE cells mature into aninterneuronal phenotype they should exhibit little spike frequencyadaptation, which is a hallmark electrical feature of GABAergicinterneurons. In current-clamp recordings from fifteen GFP⁺ cellssampled in cortical layer V, we measured mean values of −70.9±0.9 mV forresting membrane potential (RMP) and 101.4±4.1 MO for input resistance(R_(IN)). In fourteen GFP⁺ cells, depolarizing current pulses elicitedaction potentials (3.0±0.4 ms duration; 69.0±3.3 mV amplitude) andhyperpolarizing current pulses evoked a small degree of “sag” current(FIG. 5B). These intrinsic membrane properties are in the expected rangefor “mature” non-accommodating cortical interneurons (Markram et al.,(2004) Nat Rev Neurosci 5, 793-807). Most importantly, long durationdepolarizing pulses (1000 ms) clearly revealed the fast-spiking, littleadapting firing activity characteristic of basket-cell corticalinterneurons. One cell did not exhibit active firing properties duringstep depolarisations, but had a RMP of −70 mV and R_(IN) of 100 MΩ. Thehigh firing frequency typical of GFP⁺ cells sampled is shown in FIG. 5B;frequency-current relationships were linear as previously reported forfast-spiking hippocampal interneurons (FIG. 5C) (Smith et al., (1995) JNeurophysiol. 74, 650-72).

Transplanted MGE Cells Influence Synaptic Function in the Host Animal.

To determine whether transplanted MGE precursors functionally integratein the host brain, a series of in vitro electrophysiological studieswere performed. Regions of neocortex containing GFP⁺ cells wereidentified under epifluorescence (FIG. 6) and pyramidal neurons inregions surrounded by GFP⁺ cells were chosen for patch-clamp recording.Recorded cells were filled with Lucifer yellow for post hoc confirmationof cell location and identity (FIG. 6A). Brain slices were prepared atvarious time-points following transplantation (2, 3 and 4 weeks).Spontaneous IPSCs on pyramidal neurons (FIG. 7A) reflect activation ofpostsynaptic GABA receptors following action potential-dependentvesicular transmitter release; IPSCs were completely abolished by 10 μMBMI a GABA_(A) receptor antagonist (data not shown). If a significantnumber of transplanted MGE cells integrate into the host micro-circuitryas new GABAergic interneurons, we would expect an increase in theoverall level of GABA-evoked synaptic events onto native pyramidalneurons. Increments in GABA-, PV- and SOM-expressing neurons wereobserved in the cortical hemisphere ipsilateral to the injection sitewhen compared to contralateral hemisphere (Table 2). These incrementswere significant in a 100 μm area around the graft. In concordance withthese anatomical observations, there were significant increases in IPSCamplitude and frequency in slices from transplanted animals 4 weeksfollowing surgery. Control cortical slices were obtained fromsham-operated mice or from the contralateral cortex of transplanted mice(which lacked GFP⁺ cells) (FIGS. 7B-C). IPSC frequency and amplitudewere also increased in the hippocampus of grafted animals at 4 weekspost-transplantation (FIG. 8). Consistent with an increase in the numberof GABA-producing neurons, mIPSC frequencies were also increased inneocortical and hippocampal pyramidal cells 4 weeks aftertransplantation (cortex: 2.3±0.1 Hz n=4; CM: 2.4±0.2 Hz, n=3) whencompared with controls (cortex: 1.3±0.2 Hz, n=4; CA1: 1.1±0.1 Hz, n=3;p<0.05). A significant enhancement of GABAergic inhibition was notobserved at 2 or 3 weeks following transplantation; not surprisingly ashistological analysis at these times showed an immature phenotype ofgrafted cells. Significant changes in IPSC rise time or decay-timeconstant were not observed at any time-point (FIG. 76) suggesting thatgross alterations in postsynaptic GABA subunit receptor expression donot occur in grafted animals.

To assess the overall level of inhibitory tone in grafted animals, weperformed two additional analyses. First, measurement of the totalcharge transfer (corresponding to total area under the IPSC current overa specified time period) indicated that synaptic inhibition wassignificantly increased in slices containing GFP⁺ cells compared toage-matched controls (FIGS. 7C-D). Second, consistent with anenhancement of GABAergic tone, there was a significant increase in thefrequency of sIPSCs plotted as a cumulative distribution (FIG. 7E).

To test whether the transplanted MGE cells synapse onto existinginterneurons, and thereby modify cortical excitation (through inhibitionof interneuron function), EPSCs were analyzed. EPSCs recorded frompyramidal neurons (holding potential of −75 mV) in regions containingGFP⁺ cells; spontaneous EPSCs were abolished by application of CNQX andAPV confirming a role for postsynaptic glutamate receptors. In comparingspontaneous EPSCs recorded on pyramidal cells from MGE transplantedanimals (n=4) and controls (n=4) no difference in amplitude, decay-timeconstant, rise-time or frequency was noted (FIG. 9A). These findingssuggest that overall excitatory tone in the host brain is not alteredfollowing grafting of MGE precursors. To address whether transplantedneurons receive excitatory synaptic contact from host axons, we nextexamined evoked and spontaneous EPSCs in GFP⁺ neurons. GFP⁺ cellsexhibited spontaneous EPSCs that were blocked by CNQX and APV (n=4)(FIG. 9B) and evoked EPSCs with a reversal potential near 0 mV (FIG.9C). EPSCs exhibited kinetics similar to those expected for “normal”glutamate-mediated synaptic currents. These results confirm anendogenous excitatory excitation of grafted MGE-GFP neurons. Takentogether, these data suggest that MGE-derived GFP⁺ cells function asinhibitory interneurons receiving excitatory input from local pyramidalneurons and integrating into cortical synaptic circuitry of the hostbrain in such a manner as to selectively modify inhibition.

The example demonstrates that MGE-derived neuronal precursors graftedinto the early postnatal brain are capable of long distance dispersionacross the neocortex and other areas of the juvenile brain. These cellsthen acquire morphological, molecular and physiological characteristicsof mature GABAergic interneurons. Finally, these grafted MGE-derivedcells functionally integrate and significantly impact synapticinhibition in the host brain. Thus, the present example demonstratesthat MGE precursors could be used to modify synaptic circuits in apostnatal brain. An ability of these cells to disperse when transplantedinto the neonatal brain is demonstrated, reaching maximum migrationdistances of 5 mm two months after transplantation. As such, a singleinjection of MGE precursors could influence a relatively wide area ofthe host brain, an important aspect when considering the potentialclinical usefulness of transplanted cells. The present results show thatmore than 65% of MGE-derived cells express GABA. Grafted cells alsocontain SOM and NPY, neuropeptides normally co-localized in subtypes ofmature cortical interneurons (DeFelipe, (1993) supra; Kubota et al.,supra; DeFelipe, (1997) supra; Gonchar and Burkhalter, supra). We didnot detect pyramidal-like neurons or astrocytes that were derived fromtransplanted MGE cells. Importantly, tumors were never observed in ourMGE grafted mice although this is a common problem when ES-derivedprogenitors are used for transplantation (Wernig et al., supra;Ruschenschmidt et al., supra). MGE-derived cells sampled in layer Vexhibit an “electrical fingerprint” typical of mature GABA-containinginterneurons. For example, MGE-GFP cells consistently fired at a highfrequency and exhibited very little accommodation. These firingproperties are consistent with a classification as non-accommodatingbasket-cell interneurons and it is likely that further current-clampsampling of GFP⁺ cells across other layers of grafted cortex willuncover additional interneuron sub-types. In previous analysis offunctional integration, single-cell recordings focused exclusively ondemonstrations that transplanted cells receive synaptic input. Here wealso demonstrate that transplanted MGE-derived cells receive excitatorysynaptic input (see FIG. 6). Moreover, we present evidence that graftedprogenitor cells send inhibitory outputs, which impact (in afunctionally relevant manner) existing pyramidal neurons. Notably, wefound that pyramidal cells in regions containing MGE-derived cellsexhibit an increased number of GABA-mediated synaptic events and thatGABAergic tone is significantly enhanced in these regions of the hostbrain. Because MGE-derived cells did not alter excitatory corticalcircuitry or differentiate to neurons with a pyramidal-cell phenotype,these findings suggest a method for selective enhancement of inhibitorysystems.

Our demonstration that grafted progenitor cells produce functionallyintegrated GABAergic neurons, even in the presence of endogenousGABAergic neurons, after embryonic stages of neurodevelopment arecomplete, and in a wide variety of brain regions, suggests thatMGE-derived cells could be useful in neurological conditions whereincreased inhibition would be beneficial e.g., epilepsy orschizophrenia. MGE precursors may also be used to correct levels ofactivity in deafferented brain regions such as in Parkinson's disease,or in conjunction with their inhibitory function, may be used ascellular vectors to deliver therapeutic molecules to wide regions of thebrain.

TABLE 1 MGE graft derived interneuron subtypes (n = 5) GABA PV SOM CRNPY CORTEX 68.6 ± 4.8% 38.3 ± 5.4% 43.2 ± 3.9%  1.9 ± 0.6%  7.8 ± 1.2%53.1 ± 5.3%^(a) 10.3 ± 1.3%^(c) 33.2 ± 2.4%^(b) STRIATUM 50.9 ± 2.6%54.9 ± 7.6% 39.5 ± 4.6%  6.4 ± 1.9% 18.0 ± 2.1% HIPPO (DG) 42.8 ± 2.9%33.7 ± 4.7% 33.8 ± 8.1% 10.3 ± 1.7% 13.1 ± 1.9% Quantifications wereperformed in somatosensory cortex except for ^(a)Layers I-III ofsomatosensory cortex, ^(b)Layers IV-VI of somatosensory cortex, and^(c)Retrosplenial cortex. DG; Dentate Gyrus

TABLE 2 Interneuron increment in transplanted somatosensory cortexCORTEX¹ CORTEX² (100 μm) (1200 μm) GABA 12.1 ± 3.7% (P < 0.01)  8.4 ±3.5 (P < 0.01) Pv  9.8 ± 2.1% (P < 0.01)  4.8 ± 3.6% (P = 0.23) SOM(I-III) 16.1 ± 2.8% (P < 0.01) 12.9 ± 3.6% (P < 0.05) Contralateralresults were taken as 100%. ¹Estimation of cell increment 100 μm aroundof injection site. Quantification was performed in 2 slices 50 μmforward and backward from injection site. ²Estimation of cell increment1200 μm around of injection site. Quantification was performed in 3slices forward plus 3 slices backward from injection site, Significance(p) was estimated with a T-student test. N = 10.

Example 2: Robust Epileptiform Burst Activity is More Difficult toInitiate in Slices Containing MGE Progenitors

Attempts are made to elicit epileptiform burst activity in corticalslices having received MGE progenitor cell grafts and control corticalslices that have not received MGE progenitors. It is determined to bemore difficult to initiate robust epileptiform burst activity in slicescontaining MGE progenitors. This finding supports that MGE progenitorsmigrate and differentiate into functional interneurons in the host brain(and thus increase synaptic inhibition).

Neocortical slices are prepared from wild-type mice with MGE grafts andage-matched controls. Spontaneous seizure activity is initiated inneocortical slices by raising the extracellular level of potassium, in astep-wise fashion, from 3 to 6 to 9 mM [K⁺]_(e). Previous studies in ourlaboratory (Baraban and Schwartzkroin, Epilepsy Res. 1995 October;22(2):145-56) and others (Rutecki et al., J Neurophysiol. 1985 November;54(5):1363-74; Traynelis and Dingledine, J Neurophysiol. 1988 January;59(1):259-76), demonstrate this is an efficient method to inducespontaneous seizure activity and test anticonvulsant drugs in vitro. The“high K” model reliably elicits status-like interictal-like epileptiformactivity and is designed to mimic high [K⁺]_(e) observed during clinicalseizures. Epileptiform activity is monitored using field recordingelectrodes placed in outer (Layers IV/V) and inner (Layer II) neocortex.Epileptiform burst discharge amplitude (in mV), duration (in msec) andfrequency (in Hz) is used to quantitatively compare bursting betweenexperimental and control animals. A second method to compare interictal“burst intensity” in different [K⁺]_(e) involves the use of a coastlinebursting index (CBI) (Korn et al., J Neurophysiol. 1987 January;57(1):325-40). CBI is responsive to changes in the number or amplitudeof bursts, and it increases when neuronal synchrony, firing frequency orduration changes—thus, it can be considered a sensitive measure ofwhether integrated MGE progenitors influence seizure activity.

A separate series of identical experiments is performed using thezero-Mg²⁺ acute seizure model. Removal of Mg²⁺ from the extracellularbathing medium releases magnesium blockade of NMDA-type glutamatereceptors and initiates epileptiform activity driven by excess synapticexcitation (Mody et al., J Neurophysiol. 1987 March; 57(3):869-88).Epileptiform activity elicited in slices from grafted mice is comparedwith age-matched controls. Analysis is performed as described above.Slices are postfixed and immunostained with an antibody to GFP so thenumber of grafted MGE-GFP⁺ cells can be assessed. Using these twodifferent mechanisms of action we reliably determine transplantedprogenitors exert anticonvulsant action in vitro.

Results: It is determined to be more difficult to initiate robustepileptiform burst activity in slices containing MGE progenitors. Thisfinding supports that MGE progenitors migrate and differentiate intofunctional interneurons in the host brain (and thus increase synapticinhibition). A decrease in burst amplitude, duration or frequency or achange in CBI index provides quantitative evidence that integrated MGEprogenitors, by increasing inhibition, reduce epileptichyperexcitability.

Example 3: Seizures are More Difficult to Initiate in Mice Receiving MGEProgenitors

Following bilateral MGE grafting in wild-type mice (and sham operatedcontrols; young adult P30 and adult P60 ages) EEG electrodes areimplanted bilaterally in neocortex and animals monitored with video-EEG.After a 1 wk recovery period, following surgery, animals are injectedwith kainic acid (KA, a glutamate receptor agonist) at a concentrationpreviously shown to elicit status epilepticus in the mouse e.g., 30-40mg/kg i.p. (Baraban et al., Brain Res Dev Brain Res. 1997 Sep. 20;102(2):189-96; Baraban et al., J Neurosci. 1997 Dec. 1; 17(23):8927-36).In analyzing video-EEG traces following initiation of a KA-inducedseizure, the frequency and duration of electrographic seizure eventsrecorded are quantified. Behaviors that accompany these discharges arefully characterized by close examination of the video-EEG recordingsusing an investigator blind to the status of the animal.Clinician-scientists in the laboratory with significant clinical EEGexperience assist in analysis of this data.

A second set of identical experiments are performed usingpentylenetetrazole (a GABA antagonist, 15-20 mg/kg i.p.). Similar toslice electrophysiology studies, two separate means of seizure inductionare used to adequately assess the ability of MGE progenitors todecrease/inhibit seizure activity.

In all animals, euthanasia and transcardial perfusion are performed atthe conclusion of video-EEG experiments. Brains are rapidly removed andfixed in paraformaldehyde for post hoc confirmation of EEG electrodeplacement. In addition, brains are sectioned and stained for analysis ofGFP⁺ interneurons. These anatomical studies allow us to correlatenumbers of integrated GFP progenitors with antiepileptic activity.

Results: It is more difficult to initiate seizures in mice receiving MGEprogenitors. Electrographic seizure events, if observed, are brief intransplanted animals and little or no signs of convulsive behavior areobserved. Animals with large numbers of integrated MGE progenitors aremost resistant to the development of acute seizure activity.

Example 4: MGE Progenitors Reduce Seizure Activity in Mouse Models ofSpontaneous Epilepsy

Transplanted MGE progenitor cells are used to enhance synapticinhibition such that seizure susceptibility is significantly reduced inthe host animal. Studies are performed in neocortical tissue sectionsfrom wild-type control mice (following grafting) and mouse mutants withknown cortical interneuron defects. Three mutants with a demonstratedreduction in synaptic inhibition and hyperexcitability are used:particularly, Dlx1−/−, GAD65^(−/−) and uPAR^(−/−). Dlx1 mice showgeneralized electrographic seizures and histological evidence ofseizure-induced reorganization and hence display a phenotype comparableto that of human epilepsy associated with interneuron loss. GAD65mutants appear to have normal numbers of GABAergic corticalinterneurons, but a reduced capacity to synthesize GABA (Kash et al.,Proc Natl Acad Sci USA. 1997 Dec. 9; 94(25):14060-5). uPAR mutantsappear to have a reduced density of GABAergic interneurons in parietalcortex (Powell et al., J Neurosci. 2003 Jan. 15; 23(2):622-31).

(i) GAD65−/− and uPAR−/− Mice

Hyperexcitable states have been reported in mutants with abnormalcortical interneurons (GAD65 KO) and in mutants with reduced numbers ofcortical interneurons (uPAR KO). First, disruption of the GAD65 gene inmice leads to a 50% decrease in cofactor-inducible GAD enzymaticactivity (Kash et al., supra). GAD65-deficient mice on a C57BI/6background are susceptible to infrequent spontaneous seizures andstress-induced seizures. Second, inactivation of the urokinaseplasminogen activator receptor (uPAR) gene in mice leads to a 50-65%reduction in cortical GABAergic interneurons (Powell et al., supra).uPAR KO mice (bred on a C57BI/6 background) are viable, survive intoadulthood, and exhibit overt tonic-clonic seizures or an increasedsusceptibility to PTZ-induced motor convulsions. Both strains of mutantmice are used. Because background strain can be an important modulatorof seizure susceptibility (Schauwecker and Steward, Proc Natl Acad SciUSA. 1997 Apr. 15; 94(8):4103-8; Schauwecker, Prog Brain Res. 2002;135:139-48), we are careful to study mutant and wild-type mice bred ononly one background strain e.g., the relatively seizure-resistantC57BI/6.

Following bilateral MGE grafting in GAD65^(−/−) or uPAR^(−/−) mice, andsham operated, strain- and age-matched controls, EEG electrodes areimplanted bilaterally in neocortex and monitored with video-EEG. After a1 wk recovery period, following surgery, animals are monitored each dayfor 6 hr recording sessions (2 wk monitoring period). In analyzingvideo-EEG traces the frequency and duration of electrographic seizuresrecorded are quantified. Behaviors that accompany these discharges arefully characterized by close examination of video-EEG recordings usingan investigator blind to the status of the animal; clinician-scientistsin the laboratory assist in these studies. The frequency and amplitudeof interictal spikes may vary during sleep-wake cycles (Martins da Silvaet al. Electroencephalogr Clin Neurophysiol. 1984 July; 58(1):1-13). Assuch, interictal spikes are always analyzed during periods of non-REMsleep. Because mutant mice can exhibit spontaneous seizure activity(consisting of frequent abnormal slow waves and interictal dischargeswith associated convulsive behaviors) it is not necessary to induceseizures using kainate or PTZ.

We sacrifice these animals and quantify the number of new GABAergic GFP⁺interneurons present in neocortex. We correlate the number of GFP⁺ cellswith seizure severity (as determined from analysis of behavior and EEG).Detailed immunocytochemical studies using antibodies to GAD, NPY,parvalbumin, somatostatin and calbindin are performed. A limited numberof slice electrophysiology studies are also performed to analyze sIPSCsin un-treated and grafted animals.

Results: The reduction in functional GABAergic interneurons resulting ina spontaneous epileptic phenotype observed in uPAR KO mice is alleviatedby grafting MGE progenitors into these animals. Interictal spikes andbehavioral seizures are reduced (or eliminated) in uPAR KO micereceiving MGE grafts. Similar results are observed in GAD65 mutant mice.

(ii) Dlx−/− Mice

MGE cells were transplanted into the brains of Dlx1−/− mice, a murinemodel of epilepsy, in a similar manner as described in example 1. Fordetails on Dlx1−/− mice, see Cobos et al., Nature Neuroscience,8:1059-1068, 2005, expressly incorporated herein in its entirety byreference. Dlx1 mice show generalized electrographic seizures andhistological evidence of seizure-induced reorganization and hencedisplay a phenotype comparable to that of human epilepsy associated withinterneuron loss. Dlx1 mutant mice transplanted with MGE progenitorcells appeared to have a reduced epilepsy phenotype, measured as areduction in seizure-like behavior upon handling and a lack of EEG-likeseizure activity. Cortical brain slices prepared from Dlx mutant micetransplanted with MGE progenitor cells early in development (P0-P2)exhibited a level of inhibition (measured as spontaneous and miniatureIPSCs on postsynaptic pyramidal cell targets in regions containingMGE-GFP interneurons) that was comparable to that observed in controlDlx heterozygote mice. Specifically, Dlx mutants normally showed reducedIPSC frequency and amplitude and these values were “rescued” by MGEtransplantation. See FIG. 10. At the whole animal level, Dlx mutantsnormally exhibited handling induced seizures and spontaneous seizures.Dlx mutants transplanted with MGE cells did not exhibit handling inducedseizures and video-EEG recording confirmed the lack of a seizurephenotype. This demonstrated that MGE cells can be successfullytransplanted into the diseased brain and demonstrated reduction orablation of epileptic symptoms following transplantation.

Example 5: MGE Precursors Increase Seizure Latencies and ReduceMortality in a Rodent Seizure Model

A commonly used rodent seizure model (e.g., pilocarpine) was used toinvestigate the therapeutic potential of MGE-derived interneurons. MGEcells from e13.5 GFP-expressing mice were transplanted into thepostnatal (p4) brain using procedures described above. After allowingfor migration and integration to occur, single doses of scopolaminefollowed by pilocarpine (300 mg/kg) were administered to induce acuteseizure activity. Mortality and seizure latency were compared amongsham-transplanted mice, MGE cell-transplanted recipients, and micepretreated with phenobarbital (PB), a conventional AED (antiepilepticdrug).

Seizure behaviors were scored on a Racine scale by an investigator blindto the status of the animal. It was observed that the transplanted miceand PB-pretreated mice had longer seizure latencies and lower mortalityrates compared to sham-transplanted littermates. In grafted mice,seizure protection correlated with the number of newly generated MGE-GFPcells.

Immunohistochemistry and electrophysiology were then carried out asdescribed herein to confirm whether the therapeutic benefit observed inthe transplanted mice was due to the inhibitory activity of MGE-derivedinterneurons. The immunohistochemistry revealed that MGE-derivedtransplanted cells in the neocortex and hippocampus were mostly neuronal(NeuN+) and GABAergic, as expected. Whole-cell electrophysiologicalrecordings of presynaptic GFP+ cells and postsynaptic pyramidal cellsconfirmed that transplanted cells were able to functionally integrateand increase synaptic inhibition, as well as receive excitatory inputsfrom endogenous pyramidal cells.

These results indicate that MGE-derived precursor cells are able tomigrate large distances and functionally integrate into existingcortical circuitry, thereby reducing the harmful effects of inducedseizures in transplanted mice. These in vivo data provide a strongindication that MGE-derived precursor cells will have therapeutic valuein seizure disorders, and other disorders of inhibition, includingepilepsy and other disorders described herein.

All references cited are expressly incorporated herein in their entiretyby reference.

The invention claimed is:
 1. A method of reducing seizure activity in amammal with a seizure disorder, the method comprising: transplantingmedial ganglionic eminence (MGE) cells into the central nervous systemof a mammal with a seizure disorder; and allowing the transplanted cellsto migrate and integrate into the central nervous system of said mammalto form functional inhibitory interneurons that are associated with areduction of seizure activity in the mammal, thereby reducing seizureactivity in the mammal.
 2. The method of claim 1, wherein the MGE cellsare transplanted into the brain of said mammal.
 3. The method of claim1, wherein the mammal is selected from the group consisting of mouse,rat, human, livestock animal and domestic animal.
 4. The method of claim1, wherein the MGE cells are transplanted into a region of the centralnervous system selected from the group consisting of cerebral cortex,hippocampus, thalamus, and striatum.
 5. The method of claim 4, whereinthe MGE cells are transplanted into a region of the central nervoussystem which is free of lesions.
 6. The method of claim 1, wherein saidtransplanting comprises injecting dissociated MGE cells into the centralnervous system.
 7. The method of claim 6, wherein the MGE cells areinjected in association with a carrier.
 8. The method of claim 1,wherein the mammal is a human.
 9. The method of claim 1, wherein themammal is an adult.
 10. The method of claim 1, wherein the mammal is ajuvenile.
 11. The method of claim 1, wherein said seizure disorder isepilepsy.
 12. A method of increasing the seizure latency in a mammalwith a reduced capacity to synthesize γ-aminobutyric acid (GABA) in thecentral nervous system (CNS), the method comprising: transplantingmedial ganglionic eminence (MGE) cells into the central nervous systemof a mammal with a reduced capacity to synthesize GABA in the CNS; andallowing the transplanted cells to migrate and integrate into the CNS ofsaid mammal to form functional inhibitory interneurons that areassociated with an increase in GABA production in the CNS of the mammal,thereby increasing seizure latency in the mammal.
 13. The method ofclaim 12, wherein said transplanting comprises injecting dissociated MGEcells into the central nervous system.
 14. The method of claim 13,wherein the MGE cells are injected in association with a carrier. 15.The method of claim 12, wherein the mammal is a human.
 16. The method ofclaim 12, wherein the mammal is an adult.
 17. The method of claim 12,wherein said mammal suffers from epilepsy.
 18. A method of treating amammal with demonstrated increased neural hyperexcitability in thecentral nervous system (CNS), the method comprising: transplantingmedial ganglionic eminence (MGE) cells into the central nervous systemof a mammal with demonstrated increased neural hyperexcitability; andallowing the transplanted cells to migrate and integrate into the CNS ofsaid mammal to form functional inhibitory interneurons that areassociated with reduced neural hyperexcitability and improved functionin the CNS of the mammal, thereby treating the mammal.
 19. The method ofclaim 18, wherein said transplanting comprises injecting dissociated MGEcells into the central nervous system.
 20. The method of claim 19,wherein the MGE cells are injected in association with a carrier. 21.The method of claim 18, wherein the mammal is a human.
 22. The method ofclaim 18, wherein the mammal is an adult.